1 PROJECT SUMMARY 2 There remains a critical knowledge gap regarding the spatiotemporal dynamics of bacterial tissue 3 dissemination, as well as how a diverse range of bacteria broadly and differentially influence phenotypes 4 referred to as the hallmarks of cancer. As one particularly illustrative example, Fusobacterium nucleatum is 5 a resident, non-motile, Gram-negative anaerobe of the oral cavity with the ability to disseminate and cause 6 lethal infections of the brain, lungs, and liver, and is often found in abundance in colorectal cancer (CRC) 7 tissue. It is known that entry of this bacterium into host cells is a critical component in the activation of pro- 8 oncogenic pathways, yet the tissue dissemination mechanisms for F. nucleatum past initial cellular entry in 9 the oral cavity remains poorly understood. Recently it was demonstrated that this bacterium is able to 10 survive within infected host tumor cells for long enough to be carried along to distant metastatic sites such 11 as the liver. And we have more recently demonstrated that these bacteria invade CRC organoids, and 12 intriguingly localize to crypt niches of critical importance in CRC progression. These observations suggest a 13 potential role for bacteria in CRC and provide a rationale to further understand how F. nucleatum gains 14 access to normally sterile tissues. We believe that recent breakthroughs in tissue engineering will enable 15 studies that are capable of revealing the broad-ranging effects of bacteria in the tumor microenvironment 16 (TME), including but not limited to i) the dissemination dynamics through blood vessels and within the tumor 17 perivascular niche, and ii) the impacts of bacteria on CRC progression via regulation of epithelial-to- 18 mesenchymal transition (EMT) that has been implicated in the colon stem cell niche. In this highly 19 exploratory R21 project we will overcome the major limitations to such studies, which include the over- 20 proliferation of tumor and bacterial cells in vitro, and the limited spatiotemporal resolution in vivo. We will 21 leverage tissue engineering combined with microfluidic analysis and bacterial genetic technologies to 22 enable high-resolution studies of tumor-microbe interactions in an in vitro biological system modeled after 23 the human TME. We will proceed with the following aims: 1) Develop a microfluidic spheroid-based tissue 24 model capable of mimicking F. nucleatum tropism out of the circulation and into the colon TME, and 2) 25 Perform unbiased transcriptome and epigenome profiling of epithelial pathway activation resulting from F. 26 nucleatum infection under fully-defined CRC TME conditions. More broadly, this project could result in an 27 adaptable technology with higher throughput, accuracy, and cost-effectiveness compared with competing 28 methods to analyze dynamic host-pathogen interactions, help to define new roles for tumor-microbe 29 interactions in complex cancer processes, and aid in the identification and screening of future targets for 30 cancer vaccines or therapies.